Cell culturing, which is the growth of cells in an artificial in vitro environment, is a crucial technique in life science research and development and many biotechnology and health applications. An ideal cell culturing environment is one that promotes fast and robust growth of healthy cells, wherein the cell morphology and function are dominated by cell-cell interactions, cell-specific signaling, and/or experimental control variables, rather than being influenced by the properties of the artificial culturing environment. Often, it is desirable to grow cells that closely resemble cells grown in living organisms, including their gene expression, functional characteristics of differentiated cells, and the formation of an extracellular matrix. Cost and scalability of production are also critical considerations for the application potential of such technologies.
As interest in nanotechnology, materials, and cellular biology has grown, it has become evident that an important limitation in our ability to manipulate, grow and use cells and tissues has been our lack of ability to control the pattern of cells and tissues useful for cellular biology and medicine.
During development of living organisms, structure and order in the form of patterns naturally emerge through mechanisms that are still not fully understood. If one wants to study or replicate living tissue in an artificial environment, it is critical to be able to reproduce the naturally existing patterns. The ability to engineer and manually control the patterns of living cells, especially in three-dimensions and on surfaces, will enable many bioengineering and medical applications heretofore not realized.
Traditional cell culturing in Petri dishes produces two-dimensional (2D) cell growth with gene expression, signaling, and morphology that can differ significantly from conditions in 3D living organisms, and thus compromising the clinical relevancy of the cells or tissue for medical use.
While rotating bioreactors or protein-based gel environments have been developed in attempts to allow three-dimensional (3D) cell culturing, broad application of such methods has been severely hampered by high-cost or complexity. Thus, a platform technology to enable 3D cell culturing is still an unmet need.
Furthermore, as the use of cultured cells continue to develop, it is increasingly difficult to develop systems for safely manipulating and handling these entities. For example, regulatory agencies and good laboratory practices often attempt to minimize the amount of exposure of cells to external objects, so as to minimize contamination. Thus, devices which can manipulate cells and tissue without exposure to external environment is desirable.
A recent development in 3D cell culturing techniques is to use magnetic forces on cells or on magnetic microcarriers coated with cells, to create three dimensional cell cultures (e.g., Akira in US2006063252, WO2004083412, WO2004083416; Becker in US2009137018, WO2005003332; Felder in US2005054101, WO2005010162; Souza in WO2010036957; Ito, et al., Medical Application of Functionalized Magnetic Nanoparticles, JOURNAL OF BIOSCIENCE AND BIOENGINEERING 100(1): 1-11 (2005); and Souza, G. R. et al. Three-dimensional Tissue Culture Based on Magnetic Cell Levitation. Nature Nanotechnol. 5, 291-296, doi:10.1038/nnano.2010.23 (2010)).
The current state of the art in magnetic culturing devices is the simple magnet on top of a Petri dish used by Souza in WO2010036957 and Souza, et al. Three-dimensional Tissue Culture Based on Magnetic Cell Levitation. Nature Nanotechnol. 5, 291-296, doi:10.1038/nnano.2010.23 (2010). While, simple and at least effective in principle, such devices are not amenable to scale up, are easily dislodged, and do not allow for complex manipulations of culture conditions or magnetic cells.
Ito, et al., Medical Application of Functionalized Magnetic Nanoparticles, Journal of Bioscience and Bioengineering 100(1): 1-11 (2005), and US2006063252, WO2004083412, WO2004083416 merely use a neodymium magnet placed outside the bottom of the well. Like the magnet on a lid, these are not amenable to scale up, are easily dislodged, and do not allow for complex manipulations of culture conditions.
US2005054101 and WO2005010162 describes a machine for holding and moving magnets to move, position, and agitate magnetic microcarriers and attached cells. However, this device is not compatible with microscopy tools, and it requires a stand-alone complex device. Neither does it provide easy access to the levitated cultures, thus making it difficult to manipulate the cultures. Furthermore, cells have to be first attached to the surface of microcarriers, which are several times larger than a single cell. This introduces an artificial substrate with which cells interact, rather than rapidly promoting natural cell-cell interactions. The magnetic fields and field gradients produced by this arrangement are also relatively weak and require cells to be attached to large microcarriers containing a large amount of magnetic material in order to manipulate them.
US2009137018 and WO2005003332 described a static arrangement of magnets for levitation of microcarriers. This device is cumbersome however, and not suitable for scale up. Also, this device requires cells to be placed in a bag and a large magnet is above or around the large plastic bag. Thus, the device is not compatible with microscopy tools. Neither does it provide easy access to the levitated cultures, making it difficult to manipulate the cultures. Furthermore, again the cells are first attached to the surface of the microcarriers, which as discussed above introduces an artificial substrate into the culture. The magnetic fields and field gradients produced by this arrangement are also relatively weak and require cells to be attached to large microcarriers containing a large amount of magnetic material in order to manipulate them.
To make 3D cell culturing with magnetic forces more convenient, flexible, and safer for users, there is a great need for improving the methods and hardware to hold magnets in the proper orientation with respect to the cells and container in which they are contained. There is also a great need for methods and hardware for manipulating the magnets and cells during and after culturing. In many cases it would be advantageous to have such systems be compatible with commonly used cell culturing vessels like flasks and Petri dishes, multi-well plates, and high-throughput culturing systems.
Thus, what is needed in the art are magnetic culture devices and magnetic pipettes that can be used with existing robotics and microtiter or Petri plates, and that are simple, robust, easily scaled up and inexpensive. Further, the devices should be tunable for the application of interest. Thus, ideally the magnetic field shape or intensity can be easily varied in a manner that is simple, convenient, reproducible and consistent with sterile cell culture techniques.